The present invention relates to phosphatidic acid esters of aliphatic and cycloaliphatic hydroxylated hydrocarbons substituted with carboxylic functions. These compounds are particularly useful in the fields of drug targeting with liposomes and MRI contrast media for diagnostics.
Aliphatic esters of diacyl-glycerophosphatidic acid (diacyl glycerophosphate esters) are well known since some of them, like phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol, are natural phospholipid constituents of lecithins.
Synthetic esters of diacyl-glycerophosphatidic acid have been reported, for instance phosphatidyl esters of acyl and benzyl-substituted glycerol (CA 92-110494/13 and CA 94-046877/07) or an anti-retroviral diacylphosphatidic acid ester of the following hydroxy compound 
in which X is a nucleic base (van Wijk et al. Biochim. Biophys. Acta 1165 (1992), 45-52).
The present inventors have found that phosphatidic acid esters of formula 
have the unexpected useful property of stabilizing phospholipids when in admixture therewith. For instance, when phospholipids are used to make liposomes, the presence of the phosphatidic acid esters improve the entrapping capacity of the liposomic vesicles, prevents vesicle coalescence, and inhibits leakage of the entrapped substances into the liquid carrier. In the above formula, R1 and R2 are phospholipid fatty acid residues and A is an aliphatic and/or cycloaliphatic hydrocarbon chain optionally substituted by hydroxy and/or further carboxylic functions. In a preferred embodiment xe2x80x94Axe2x80x94xe2x95x90xe2x80x94(CH2)nxe2x80x94 where n is an integer of from 3 to about 14, preferably 5 to 10.
The fatty acid residues R1 and R2 are preferably derived from saturated or unsaturated fatty acids commonly found in lecithins, for instance the acids lauric, myristic, palmitic, stearic, arachidic, oleic, linoleic, and the like.
According to a first method of preparation of compounds of formula 
one can esterify a phosphatidic acid (II) with an acid-alcohol HOxe2x80x94Axe2x80x94COOH, for instance HOxe2x80x94(CH2)nxe2x80x94COOH (III), the acidic function of which is temporarily protected, for instance benzylated, and subsequent removal of the protective group; this is summarized in the following exemplifying scheme: 
Benzylation of the acid alcohol (III) can be effected by reacting benzyl bromide with an alcali metal salt of (III) in DMF or hexamethyl phosphoramide (HMPT) as follows: 
Of course this method is general and applies to other compounds of formula I using other acid-alcohols HOxe2x80x94Axe2x80x94COOH, the above embodiment scheme being for illustration only.
According to another route to prepare (Ia), one can react a bromo-acid Brxe2x80x94(CH2)nxe2x80x94COOH (IV) with the silver salt of the phosphatidic acid (II) 
According to still another route, on can protect one of the free xe2x80x94OH of the phosphatidic acid (II), and thereafter react the product with the bromide (V) to give the compound (VI); the latter provides the acid-ester (IA) after deprotection by usual means. This is schematized as follows: 
The direct reaction of the phosphatidic acid (II) with the bromide (V) is also possible as follows: 
Of course, the above embodiments are not limiting, the disclosed methods being general and applicable using other intermediates of formulae Brxe2x80x94Axe2x80x94COOH and Brxe2x80x94Axe2x80x94COOBzl.
The new compounds disclosed are very useful to stabilize liposome vesicles in suspension in water, buffers and biological liquids against rupture and coalescence with time. They also increase their encapsulating capacity (enhanced weight ratio of encapsulated substance versus phospholipids making the vesicles) and stabilise the vesicle membrane against leakage of the entrapped substances toward the carrier liquid. For instance, in the case where liposome vesicles are used to carry iodinated x-ray opacifying media in the organism via the blood stream, incorporating a proportion of one or more of the novel compounds to the phospholipids making the liposomes will strongly decrease the tendency of the encapsulated iodine to be released into the blood by permeating the vesicle membrane; the efficiency of transport of the opacifying compounds to possibly remote organs in the body is thus markedly increased. Usually the effective proportion of the novel compounds in the liposome forming lipids is in the range of 1 to 25% by weight, but these values can be overcome if desired. The liposome forming lipids or mixture of lipids substantially include all compounds commonly used in the field of liposomes, i.e. glycerophospholipids, non-phosphorylated glycerides, glycolipids, sterols and other additives intended to impart modified properties to liposomic membranes. Preferably, they comprise at least a polarizable component, namely a cationic or anionic function carrying lipid or an ionizable surfactant such as a fatty alcohol diphosphate ester, e.g. dicetyl phosphate (DCP) or a higher alkyl amine like stearylamine (SA). Charged phospholipids, i.e. fatty acid glycerides phosphatides like phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) from natural sources or synthetic (such as dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidyl glycerol (DPPG), etc.) are convenient polarizable lipid components. The glycerophospholipids may include for instance dipalmitoyl-phosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE) and the corresponding distearoyl- and dimyristyl-phosphatidylcholine and -ethanolamine (DSPC; DSPE; DMPC and DMPE). The liposome forming phospholipids may also include natural phospholipids which have been subjected to more or less extensive hydrogenation, for instance egg and soy phosphatidylcholine.
The glycolipids may include cerebrosides, galactocerebrosides, glucocerebrosides, sphingomyelins, sulfatides and sphingolipids derivatized with mono-, di- and trihexosides. The sterols, which should be used with parsimony, as too much may impede membrane permeation, encompass cholesterol, ergosterol, coprostanol, cholesterol esters such as hemisuccinate (CHS), tocopherol esters and the like.
It has also been found that freeze dried phospholipid powders comprising phosphatidyl compounds of the invention may conveniently be stored for prolonged periods of time without loss of properties. This is particularly useful when these phospholipids are used for the manufacture of therapeutic or contrast agents or are incorporated in drug delivery vehicles that may further include targeting vectors.
Liposomes produced from the freeze dried powders containing the phosphatidyl compounds of the invention have shown the same improved stability and entrapping capacity observed for the liposomes prepared in the usual way from non freeze dried formulations.
The following Examples illustrate the invention in more details: